Fig 1.
SADS-CoV oral infection leads to a significant increase of bile acids in the small intestine of piglets.
(A) Kinetic of viral shedding in fecal swabs from sucking piglets orally infected with SADS-CoV (n = 6). (B) Principal components analysis (PCA) of the duodenum and ileum from SADS-CoV-infected and mock-infected piglets. (C) KEGG analysis of small intestinal metabolites in SADS-CoV-infected and mock-infected piglets. (D) Volcano plot of small intestinal metabolites in small intestine of SADS-CoV-infected and mock-infected animals. (E) Heatmap of small intestinal metabolites in the duodenum and ileum. (F) Small intestinal bile acid (BA) concentrations of SADS-CoV-infected and mock-infected piglets. P values were determined by unpaired two-tailed Student’s t test. *: p < .05; **: p < .01; ***: p < .001; ns, not significant.
Fig 2.
Porcine intestinal enteroid (PIE) cultures support SADS-CoV replication.
(A) Ileal PIE monolayers were incubated with medium or SADS-CoV-GFP at the indicated MOIs on a rotary shaker for 1 h at 37°C. The incubated monolayers were washed three times with PBS and harvested at 1 or 48 hpi. Supernatant RNA was extracted and the number of SADS-CoV-GFP genome copies was determined by RT-qPCR and viral titer was determined by a standard TCID50 assay. (B) Cytopathic effect (CPE) was observed in ileal PIEs infected with SADS-CoV at 48 hpi (scale bar, 50 μm). (C) Immunofluorescence assay (IFA) was performed using rabbit anti-SADS-CoV-N polyclonal antibody and Alexa Fluor 594-conjugated anti-rabbit IgG as secondary antibody, and nuclei (blue) were visualized by DAPI (scale bar, 50 μm). (D) Colocalization of SADS-CoV-GFP and cellular markers (red) including E-cadherin (epithelial tight junction), Ki-67 (proliferating cells), villin (enterocytes) and chromogranin A (enteroendocrine cells), and nuclei (blue) stained by DAPI (scale bar, 50 μm). (E) Duodenal, jejunal and ileal PIE monolayers were incubated with SADS-CoV-GFP at MOI = 0.1 and the SADS-CoV-GFP replication kinetic was determined by RT-qPCR or TCID50. Data are from three independent experiments. P values were determined by unpaired two-tailed Student’s t test. *: p < .05; **: p < .01; ***: p < .001; ****: p < .0001; ns, not significant.
Fig 3.
Bile acids (BAs) promote SADS-CoV replication and affect the early stage of viral infection in PIEs.
(A) Schematic illustration of BA treatment in SADS-CoV-GFP infection. The image was created using the website https://app.biorender.com/. (B) Ileal PIE monolayers were inoculated with SADS-CoV-GFP at MOI = 0.1 alone or simultaneously with BAs for 48 h at 37°C. All BAs were added to a final concentration of 100 μM, and propionate control to 1 mM. Viral genomic copies and titer were quantified by RT-qPCR and TCID50. (C) PIE monolayers infected with SADS-CoV-GFP for 48 h in the presence of different concentrations of cholic acid (CA). (D) Colocalization of SADS-CoV-GFP (green) and SADS-CoV-N protein (red) in CA-treated PIEs and non-treated (NT) controls. (E) The growth kinetics of SADS-CoV-GFP in the presence or absence of 100 μM CA in PIEs. (F) Schematic showing various time periods of CA treatment (black arrows) during infection of PIEs. (G) Virus replication was quantified by RT-qPCR at 1 and 12 hpi. Data are from three independent experiments; P values were determined by unpaired two-tailed Student’s t test. *: p < .05; **: p < .01; ***: p < .001; ns, not significant.
Fig 4.
Bile acid (BA)-associated enhancement of SADS-CoV replication is not dependent on BA receptor signaling, innate immune regulation or viral binding.
Porcine intestinal enteroid (PIE) monolayers were infected with SADS-CoV-GFP at an MOI = 0.1 for 48 h. (A) TGR5 agonist INT-777 or (B) FXR agonist INT-747 was added to medium during and post-infection at the indicated concentrations. PIE monolayers were pretreated with (C) TGR5 antagonist triamterene or (D) FXR antagonist guggulsterone for 2 h, and then antagonists and cholic acid (CA) were added to the medium at the indicated concentrations during and after SADS-CoV-GFP infection for 48 h. (E) Expression of innate immune-associated antiviral response genes in PIE monolayers infected with SADS-CoV-GFP in the presence or absence of CA at 6 and 24 hpi. Gene expression was determined by qRT-PCR. (F) PIE monolayers were incubated with SADS-CoV-GFP or simultaneously with CA for the indicated time points at 4°C. Viral genome copies were quantified from unwashed cells to determine the amount of input virus and from washed cells to determine the amount of cell-attached virus. Data are reported as the percent of viral genomes remaining cell-associated compared with input. Data are from three independent experiments.
Fig 5.
Bile acid (BA) promotion of SADS-CoV replication is dependent on lipid rafts.
(A) Porcine intestinal enteroid (PIE) monolayers were pretreated with MβCD for 1 h, and then MβCD and cholic acid (CA) were added to the medium at the indicated concentration during and after SADS-CoV-GFP infection for 48 h. (B) PIE monolayers were pretreated with 1 mM MβCD for 1 h and supplemented with 1 mM cholesterol for 1 h, and then infected with SADS-CoV-GFP, CA and cholesterol were present during and post SADS-CoV-GFP infection. MβCD and CA-treated monolayers without cholesterol replenishment were set up as control. (C) PIE monolayers were treated with either medium alone, 100 μM CA, 100 μM UDCA or 1 mM propionate. Endocytic vesicles (red) were labeled by FM1-43FX and nuclei (blue) were visualized by DAPI (scale bar, 10 μm). Images were acquired by an LSM880 confocal laser-scanning microscope (Zeiss). Data are from three independent experiments; P values were determined by unpaired two-tailed Student’s t test. *: p < .05; **: p < .01; ns, not significant.
Fig 6.
Bile acid (BA) enhances SADS-CoV entry via caveolae-mediated endocytosis.
Porcine intestinal enteroid (PIE) monolayers infected with SADS-CoV-GFP in the presence of (A) chlorpromazine (CME inhibitor), (B) amiloride (macropinocytosis inhibitor), (C) nystatin (CavME inhibitor) or (D) dynasore (dynamin 2 inhibitor) at the indicated concentration and harvested at 48 hpi. Chlorpromazine, amiloride, nystatin and dynasore were added during and post-infection; data are from three independent experiments. (E) Caveolin-1 immunogold electron microscopy of SADS-CoV entry. Caveolin-1 was labeled with 10-nm immunogold, as indicated by the green arrow; SADS-CoV is indicated by the blue arrow (scale bar, 100 nm). P values were determined by unpaired two-tailed Student’s t test. *: p < .05; **: p < .01; ***: p < .001; ns, not significant.
Fig 7.
Bile acid (BA) enhances SADS-CoV replication through endosomal acidification.
Porcine intestinal enteroid (PIE) monolayers were pretreated with (A) NH4Cl and (B) bafilomycin A1 at the indicated concentrations for 1 h before infection, and harvested at 48 hpi. NH4Cl and bafilomycin A1 were supplemented during and post-infection. (C) PIE monolayers were treated with either medium alone, 100 μM CA, 100 μM cholic acid (CA) plus 30 mM NH4Cl or 100 μM CA plus 200 nM bafilomycin A1 for 1 h, washed with PBS three times and incubated with 200 nM LysoTracker for 30 min at 37°C (scale bar, 10 μm). Images were collected on an LSM880 confocal laser-scanning microscope (Zeiss). Data are from three independent experiments. P values were determined by unpaired two-tailed Student’s t test; *: p < .05; **: p < .01; ***: p < .001; ns, not significant.
Fig 8.
Bile acid (BA) treatment alters the trafficking dynamics of SADS-CoV along the endo-lysosomal system.
(A) Porcine intestinal enteroid (PIE) monolayers treated with medium alone or 100 μM cholic acid (CA) for 1 h. Images were acquired by confocal laser-scanning microscopy, detecting Rab5, Rab7 and LAMP1 (green), and nuclei (blue) were visualized by DAPI (scale bar, 10 μm). (B) PIE monolayers were infected with R18-SADS-CoV (red) at an MOI = 20 in the presence or absence of 100 μM CA and incubated at 37°C for 5, 15, 30 or 60 min. The cells were immunostained with Rab5, Rab7, or LAMP1 (green), and nuclei (blue) were visualized by DAPI (scale bar, 5 μm). Images were collected on an LSM880 confocal laser-scanning microscope (Zeiss). Pearson’s correlation coefficient analysis was carried out using Image J software. PIE monolayers were pretreated with cathepsin inhibitor (C) E-64 or (D) teicoplanin for 2 h, and then inhibitors and CA were added to the medium at the indicated concentrations during and after SADS-CoV-GFP infection for 48 h.
Fig 9.
Proposed model for SADS-CoV infection in porcine intestinal enteroids (PIEs) enhanced by bile acids (BA).
1) SADS-CoV infection induces a significant increase of BA concentration in the small intestine of piglets, and this BA-rich microenvironment promotes SADS-CoV replication in the epithelial cells. 2) BAs enhance SADS-CoV entry via CavME, and dynamin-2 is required for this effect. In the presence of BAs, 3) endosomal acidification is increased; 4) Rab7 expression is upregulated and 5) LAMP1 expression is downregulated. These cellular changes induced by BAs might alter the dynamics of endosomal/lysosomal system and 6) aid SADS-CoV escape into the cytoplasm. The image was created using the website https://app.biorender.com/.